Neurotransmitter-related Gene Expression Research Articles

Enrofloxacin exposure undermines gut health and disrupts neurotransmitters along the microbiota-gut-brain axis in zebrafish

The environmental prevalence of antibiotic residues poses a potential threat to gut health and may thereby disrupt brain function through the microbiota-gut-brain axis. However, little is currently known about the impacts of antibiotics on gut health and neurotransmitters along the microbiota-gut-brain axis in fish species. Taking enrofloxacin (ENR) as a representative, the impacts of antibiotic exposure on the gut structural integrity, intestinal microenvironment, and neurotransmitters along the microbiota-gut-brain axis were evaluated in zebrafish in this study. Data obtained demonstrated that exposure of zebrafish to 28-day environmentally realistic levels of ENR (6 and 60 μg/L) generally resulted in marked elevation of two intestinal integrity biomarkers (diamine oxidase (DAO) and malondialdehyde (MDA), upregulation of genes that encode inter-epithelial tight junction proteins, and histological alterations in gut as well as increase of lipopolysaccharide (LPS) in plasma, indicating an evident impairment of the structural integrity of gut. Moreover, in addition to significantly altered neurotransmitters, markedly higher levels of LPS while less amount of two short-chain fatty acids (SCFAs), namely acetic acid and valeric acid, were detected in the gut of ENR-exposed zebrafish, suggesting a disruption of gut microenvironment upon ENR exposure. Along with corresponding changes detected in gut, significant disruption of neurotransmitters in brain indicated by marked alterations in the contents of neurotransmitters, the activity of acetylcholin esterase (AChE), and the expression of neurotransmitter-related genes were also observed. These findings suggest exposure to environmental antibiotic residues may impair gut health and disrupt neurotransmitters along the microbiota-gut-brain axis in zebrafish. Considering the prevalence of antibiotic residues in environments and the high homology of zebrafish to other vertebrates including human, the risk of antibiotic exposure to the health of wild animals as well as human deserves more attention.

Nano polystyrene induced changes in anxiety and learning behaviour are mediated through oxidative stress and gene disturbance in mouse brain regions

It is widely reported now that nanoplastic particles have potential neurotoxic effects and may disturb central nervous system (CNS) function. However, the mechanism behind these toxic effects still needs to be elucidated. In the current study, we investigated the effects of polystyrene nanoplastics (PS-NPs) on changes in learning, memory, and anxiety-related behavior in mice based on some selected biochemical, molecular, and histopathological changes in three important brain regions (Cortex, Hypothalamus, and Hippocampus). Male mice were orally administered daily with two doses of 50 nm PS-NPs (0.2 mg/ml and 1 mg/ml) for 8 weeks. We observed decreased expression of neurotransmitter-related genes (VAChT, GAD, and SYP) in the cortex, hypothalamus, and hippocampus areas of the mouse brain. Other biochemical variables including, antioxidant enzymes, biomarkers for oxidative stress, and acetylcholinesterase activity showed significant alterations in all three brain regions. Molecular and neurochemical data thus suggest significant neurobehavioral changes following sub-chronic exposure to PS-NPs which may lead to enhanced anxiety-related and spatial learning and memory-related impairments by affecting limbic areas of the brain.

Photoaged microplastics induce neurotoxicity associated with damage to serotonergic, glutamatergic, dopaminergic, and GABAergic neuronal systems in Caenorhabditis elegans

Microplastics (MPs) are ubiquitous environmental contaminants that cause neurotoxicity in various organisms. MPs are typically affected by light irradiation and undergo photoaging. However, the neurotoxic effects of photoaged polystyrene (P-PS) and its underlying mechanisms remain unclear. In this study, locomotion behaviors, neuronal development, neurotransmitter levels, and the expression of neurotransmission-related genes were investigated in Caenorhabditis elegans exposed to P-PS at environment-relevant concentrations (0.1–100 μg/L). The characterization results showed that photoaging accelerated the aging process and changed the physicochemical properties of the MPs. The toxicity results suggested that exposure to 1–100 μg/L P-PS caused more severe neurotoxicity than virgin polystyrene (V-PS) with endpoints of head thrashes, body bends, wavelength, and mean amplitude. Exposure to P-PS also altered the fluorescence intensity and neurodegeneration percentage of serotonergic, glutamatergic, dopaminergic, and aminobutyric acid (GABA) in transgenic nematodes. Similarly, significant reductions in the levels of these neurotransmitters were also observed. Based on Pearson's correlation, locomotion behaviors were negatively correlated with the neurotransmission of serotonin, glutamate, dopamine, and GABA. Further investigation suggested that the expression of neurotransmitter-related genes (e.g., tph-1, eat-4, and unc-46) was significantly altered in the nematodes. Collectively, the neurotoxic effects of P-PS were attributed to abnormal neurotransmission. This study highlights the potential toxicity of MPs photoaged under environmentally relevant conditions.

Carboxyl-modified polystyrene microplastics induces neurotoxicity by affecting dopamine, glutamate, serotonin, and GABA neurotransmission in Caenorhabditis elegans

Microplastics (MPs) are ubiquitous in various environmental media and have potential toxicity. However, the neurotoxicity of carboxyl-modified polystyrene microplastics (PS-COOH) and their mechanisms remain unclear. In this study, Caenorhabditis elegans was used as a model to examine the neurotoxicity of polystyrene microplastic (PS) and PS-COOH concentrations ranging from 0.1 to 100 μg/L. Locomotion behavior, neuron development, neurotransmitter level, and neurotransmitter-related gene expression were selected as assessment endpoints. Exposure to low concentrations (1 μg/L) of PS-COOH caused more severe neurotoxicity than exposure to pristine PS. In transgenic nematodes, exposure to PS-COOH at 10–100 μg/L significantly increased the fluorescence intensity of dopaminergic, glutamatergic, serotonergic, and aminobutyric acid (GABA)ergic neurons compared to that of the control. Further studies showed that exposure to 100 μg/L PS-COOH can significantly affect the levels of glutamate, serotonin, dopamine, and GABA in nematodes. Likewise, in the present study, the expression of genes involved in neurotransmission was altered in worms. These results suggest that PS-COOH exerts neurotoxicity by affecting neurotransmission of dopamine, glutamate, serotonin, and GABA. This study provides new insights into the underlying mechanisms and potential risks associated with PS-COOH.

Neurotoxic impact of acute TiO2 nanoparticle exposure on a benthic marine bivalve mollusk, Tegillarca granosa

The release of nanoparticles (NPs) into the ocean inevitably poses a threat to marine organisms. However, to date, the neurotoxic effects of NPs remains poorly understood in marine bivalve species. Therefore, in order to gain a better understanding of the physiological effects of NPs, the impact of acute (96 h) TiO2 NP exposure on the in vivo concentrations of three major neurotransmitters, the activity of AChE, and the expression of neurotransmitter-related genes was investigated in the blood clam, Tegillarca granosa. The obtained results showed that the in vivo concentrations of the three tested neurotransmitters (DA, GABA, and ACh) were significantly increased when exposed to relatively high doses of TiO2 NPs (1 mg/L for DA and 10 mg/L for ACh and GABA). Additionally, clams exposed to seawater contaminated with TiO2 NP had significantly lower AChE activity. In addition, the expression of genes encoding modulatory enzymes (AChE, GABAT, and MAO) and receptors (mAChR3, GABAD, and DRD3) for the neurotransmitters tested were all significantly down-regulated after TiO2 NP exposure. Therefore, this study has demonstrated the evident neurotoxic impact of TiO2 NPs in T. granosa, which may have significant consequences for a number of the organism’s physiological processes.

Down-Regulation of Hippocampal Genes Regulating Dopaminergic, GABAergic, and Glutamatergic Function Following Combined Neonatal Phencyclidine and Post-Weaning Social Isolation of Rats as a Neurodevelopmental Model for Schizophrenia.

Background:Dysfunction of dopaminergic, GABAergic, and glutamatergic function underlies many core symptoms of schizophrenia. Combined neonatal injection of the N-methyl-D-aspartate (NMDA) receptor antagonist, phencyclidine (PCP), and post-weaning social isolation of rats produces a behavioral syndrome with translational relevance to several core symptoms of schizophrenia. This study uses DNA microarray to characterize alterations in hippocampal neurotransmitter-related gene expression and examines the ability of the sodium channel blocker, lamotrigine, to reverse behavioral changes in this model.Methods:Fifty-four male Lister-hooded rat pups either received phencyclidine (PCP, 10mg/kg, s.c.) on post-natal days (PND) 7, 9, and 11 before being weaned on PND 23 into separate cages (isolation; PCP-SI; n = 31) or received vehicle injection and group-housing (2–4 per cage; V-GH; n = 23) from weaning. The effect of lamotrigine on locomotor activity, novel object recognition, and prepulse inhibition of acoustic startle was examined (PND 60–75) and drug-free hippocampal gene expression on PND 70.Results:Acute lamotrigine (10–15mg/kg i.p.) reversed the hyperactivity and novel object recognition impairment induced by PCP-SI but had no effect on the prepulse inhibition deficit. Microarray revealed small but significant down-regulation of hippocampal genes involved in glutamate metabolism, dopamine neurotransmission, and GABA receptor signaling and in specific schizophrenia-linked genes, including parvalbumin (PVALB) and GAD67, in PCP-SI rats, which resemble changes reported in schizophrenia.Conclusions:Findings indicate that alterations in dopamine neurotransmission, glutamate metabolism, and GABA signaling may contribute to some of the behavioral deficits observed following PCP-SI, and that lamotrigine may have some utility as an adjunctive therapy to improve certain cognitive deficits symptoms in schizophrenia.

Modest Long‐Term Ethanol Consumption Affects Expression of Neurotransmitter Receptor Genes in the Rat Nucleus Accumbens

Over 100million people worldwide are affected by alcohol use disorders. These conditions usually take years to develop where an initial, voluntary consumption is gradually replaced by a compulsive intake of alcohol. The exact mechanisms behind this transition remain unknown. However, ethanol (EtOH) is known to interact with several neurotransmitters and receptors in the central nervous system, and chronic EtOH consumption causes alterations in these neurotransmitter systems, proposed to contribute to the development of dependence. This study aimed to repeat previous findings that animals after long-term voluntary EtOH consumption spontaneously increase their intake. That the initial encounter with EtOH causes an elevation of dopamine in the nucleus accumbens (nAc), inducing feelings of well-being and creating an incentive to continue the behavior, has been repeatedly reported in both animals and humans. The effects of chronic EtOH consumption on this region are not as well investigated. We examined both long-term EtOH consumption behavior and its consequences on expression of neurotransmitter-related genes in the nAc of the Wistar rat using quantitative polymerase chain reaction. In general, the EtOH consumption of the animals in this study was modest with an average intake of 0.9g/kg/d, and only 1 of the 24 rats consuming EtOH for 10months drastically increased its intake in line with the results of Wolffgramm and Heyne (1995). Expression of the genes for dopamine receptor 2, μ-opioid receptor, and somatostatin receptor 4 were down-regulated in animals after 2 and/or 4, but not 10, months of EtOH consumption. Chronic consumption even of modest amounts of alcohol seems to affect regulation of expression of these genes, possibly leading to changes in neurotransmitter signaling. Studies are ongoing to investigate whether these alterations are specific for the nAc.

Androgens coordinate neurotransmitter‐related gene expression in male whiptail lizards

Sex steroid hormones coordinate neurotransmitter systems in the male brain to facilitate sexual behavior. Although neurotransmitter release in the male brain has been well documented, little is known about how androgens orchestrate changes in gene expression of neurotransmitter receptors. We used male whiptail lizards (Cnemidophorus inornatus) to investigate how androgens alter neurotransmitter-related gene expression in brain regions involved in social decision making. We focused on three neurotransmitter systems involved in male-typical sexual behavior, including the N-methyl-d-aspartate (NMDA) glutamate receptor, nitric oxide and dopamine receptors. Here, we show that in androgen-treated males, there are coordinated changes in neurotransmitter-related gene expression. In androgen-implanted castrates compared with blank-implanted castrates (control group), we found associated increases in neuronal nitric oxide synthase gene expression in the nucleus accumbens (NAcc), preoptic area and ventromedial hypothalamus, a decrease of NR1 gene expression (obligate subunit of NMDA receptors) in the medial amygdaloid area and NAcc and a decrease in D1 and D2 dopamine receptor gene expression in the NAcc. Our results support and expand the current model of androgen-mediated gene expression changes of neurotransmitter-related systems that facilitate sexual behavior in males. This also suggests that the proposed evolutionarily ancient reward system that reinforces sexual behavior in amniote vertebrates extends to reptiles.

Krüppel-like Factor 11 Differentially Couples to Histone Acetyltransferase and Histone Methyltransferase Chromatin Remodeling Pathways to Transcriptionally Regulate Dopamine D2 Receptor in Neuronal Cells

The importance of Krüppel-like factor (KLF)-mediated transcriptional pathways in the biochemistry of neuronal differentiation has been recognized relatively recently. Elegant studies have revealed that KLF proteins are important regulators of two major molecular and cellular processes critical for neuronal cell differentiation: neurite formation and the expression of neurotransmitter-related genes. However, whether KLF proteins mediate these key processes in a separate or coordinated fashion remains unknown. Moreover, knowledge on the contribution of chromatin dynamics to the biochemical mechanisms utilized by these proteins to perform their function is absent. Here we report the characterization of two antagonistic, chromatin-mediated mechanisms by which KLF11, also known as TIEG2 (transforming growth factor-β-inducible early gene 2) and MODY VII (maturity onset diabetes of the young VII), regulates transcription of the fopamine D2 receptor (Drd2) gene. First, KLF11 activates transcription by binding to a distinct Sp-KLF site within the Drd2 promoter (-98 to -94) and recruiting the p300 histone acetyltransferase. Second, Drd2 transcriptional activation is partially antagonized by heterochromatin protein 1 (HP1), the code reader for histone H3 lysine 9 methylation. Interestingly, KLF11 regulates neurotransmitter receptor gene expression in differentiating neuronal cell populations without affecting neurite formation. Overall, these studies highlight histone methylation and acetylation as key biochemical mechanisms modulating KLF-mediated neurotransmitter gene transcription. These data extend our knowledge of chromatin-mediated biochemical events that maintain key phenotypic features of differentiated neuronal cells.

Si-RNA inhibition of brain insulin or insulin-like growth factor receptors causes developmental cerebellar abnormalities: relevance to fetal alcohol spectrum disorder

BackgroundIn experimental models of fetal alcohol spectrum disorder (FASD), cerebellar hypoplasia and hypofoliation are associated with insulin and insulin-like growth factor (IGF) resistance with impaired signaling through pathways that mediate growth, survival, plasticity, metabolism, and neurotransmitter function. To more directly assess the roles of impaired insulin and IGF signaling during brain development, we administered intracerebroventricular (ICV) injections of si-RNA targeting the insulin receptor, (InR), IGF-1 receptor (IGF-1R), or IGF-2R into postnatal day 2 (P2) Long Evans rat pups and examined the sustained effects on cerebellar function, structure, and neurotransmitter-related gene expression (P20).ResultsRotarod tests on P20 demonstrated significant impairments in motor function, and histological studies revealed pronounced cerebellar hypotrophy, hypoplasia, and hypofoliation in si-InR, si-IGF-1R, and si-IGF-2R treated rats. Quantitative RT-PCR analysis showed that si-InR, and to a lesser extent si-IGF-2R, broadly inhibited expression of insulin and IGF-2 polypeptides, and insulin, IGF-1, and IGF-2 receptors in the brain. ELISA studies showed that si-InR increased cerebellar levels of tau, phospho-tau and β-actin, and inhibited GAPDH. In addition, si-InR, si-IGF-1R, and si-IGF-2R inhibited expression of choline acetyltransferase, which mediates motor function. Although the ICV si-RNA treatments generally spared the neurotrophin and neurotrophin receptor expression, si-InR and si-IGF-1R inhibited NT3, while si-IGF-1R suppressed BDNF.Conclusionsearly postnatal inhibition of brain InR expression, and to lesser extents, IGF-R, causes structural and functional abnormalities that resemble effects of FASD. The findings suggest that major abnormalities in brains with FASD are mediated by impairments in insulin/IGF signaling. Potential therapeutic strategies to reduce the long-term impact of prenatal alcohol exposure may include treatment with agents that restore brain insulin and IGF responsiveness.

Region‐specific gene expression in early postnatal mouse thalamus

Previous studies in the developing mouse thalamus have demonstrated that regional identity is established during early stages of development (Suzuki-Hirano et al. J. Comp. Neurol. 2011;519:528-543). However, the developing thalamus often shows little resemblance to the anatomical organization of the postnatal thalamus, making it difficult to identify genes that might mediate the organization of thalamic nuclei. We therefore analyzed the expression pattern of genes that we have identified as showing regional expression in embryonic thalamus on postnatal days (P) 6-8 by using in situ hybridization. We also identified several genes expressed only in the postnatal thalamus with restricted expression in specific nuclei. We first demonstrated the selective expression of neurotransmitter-related genes (vGlut2, vGAT, D2R, and HTR2C), identifying the neurotransmitter subtypes of cells in this region, and we also demonstrated selective expression of additional genes in the thalamus (Steel, Slitrk6, and AI852580). In addition, we demonstrated expression of genes specific to somatosensory thalamic nuclei, the ventrobasal posterior nuclei (VP); a visual thalamic nucleus, the dorsal lateral geniculate nucleus (dLGN); and an auditory thalamic nucleus, the medial geniculate body (MGB) (p57Kip, Nr1d1, and GFRα1). We also identified genes that are selectively expressed in multiple different nuclei (Foxp2, Chst2, and EphA8). Finally, we demonstrated that several bone morphogenetic proteins (BMPs) and their inhibitors are expressed in the postnatal thalamus in a nucleus-specific fashion, suggesting that BMPs play roles in the postnatal thalamus unrelated to their known role in developmental patterning. Our findings provide important information for understanding the mechanisms of nuclear specification and connectivity during development, as well as their maintenance in adult thalamus.

Glial-Derived Neurotrophic Factor Modulates Enteric Neuronal Survival and Proliferation Through Neuropeptide Y

Glial-derived neurotrophic factor (GDNF) promotes the survival and proliferation of enteric neurons. Neuropeptide Y (NPY) is an important peptide regulating gastrointestinal motility. The role of NPY on the survival and proliferation of enteric neurons is not known. We examined the effects of GDNF on the expression and release of NPY from enteric neurons and the role of NPY in promoting enteric neuronal proliferation and survival. Studies were performed in primary enteric neuronal cultures and NPY knockout mice (NPY(-/-)). GDNF-induced expression of NPY was assessed by reverse-transcription polymerase chain reaction (RT-PCR), immunocytochemistry, and enzyme-linked immunosorbent assay. Using NPY-siRNA and NPY-Y1 receptor antagonist, we examined the role of NPY in mediating the survival and proliferation effects of GDNF. Gastrointestinal motility was assessed by measuring gastric emptying, intestinal transit, and isometric muscle recording from intestinal muscle strips. GDNF induced a significant increase in NPY messenger RNA and protein expression in primary enteric neurons and the release of NPY into the culture medium. NPY (1 mumol/L) significantly increased proliferation of neurons and reduced apoptosis. In the presence of NPY-siRNA and NPY-Y1 receptor antagonist or in enteric neurons cultured from NPY(-/-) mice, GDNF-mediated neuronal proliferation and survival was reduced. NPY increased the phosphorylation of Akt, a downstream target of the PI-3-kinase pathway. In NPY(-/-) mice, there were significantly fewer nNOS-containing enteric neurons compared with wild-type (WT) mice. NPY(-/-) mice had accelerated gastric emptying and delayed intestinal transit compared with WT mice. We demonstrate that NPY acts as an autocrine neurotrophic factor for enteric neurons.

Ganglion-Specific Patterns of Diabetes-Modulated Gene Expression Are Established in Prevertebral and Paravertebral Sympathetic Ganglia Prior to the Development of Neuroaxonal Dystrophy

In both humans and animal models, diabetic sympathetic autonomic neuropathy is associated with the selective development of markedly enlarged distal axons and nerve terminals (neuroaxonal dystrophy, NAD). NAD occurs in the prevertebral superior mesenteric and celiac ganglia (SMG-CG), but not in the paravertebral superior cervical ganglion (SCG). To identify molecular differences between these ganglia that may explain their selective vulnerability to NAD, we have examined global gene expression patterns in control and diabetic rat sympathetic ganglia before and after the onset of structural evidence of NAD. As predicted, major differences in transcriptional profiles exist between SCG and SMG-CG in normal young adult animals including, but not limited to, known differences in neurotransmitter-related gene expression. Gene expression patterns of diabetic SMG-CG and SCG, prior to the development of NAD lesions, also differ from their age-matched non-diabetic counterparts. However, diabetes has ganglion-specific effects on gene expression; of approximately 110 transcripts that were differentially expressed between diabetic and control sympathetic ganglia, only 5 were differentially expressed as a result of diabetes in both SCG and SMG-CG. Genes involving synapse and mitochondrial structure and function, oxidative stress, and glycolysis were highly represented in the differentially expressed gene set. Differences in the number of synapse-related gene alterations in diabetic SMG-CG (18 genes) versus SCG (2 genes) prior to the onset of NAD may also well explain the selective development of NAD in the SMG-CG. These results provide support for the specificity of diabetes-modulated gene expression for selected neuronal subpopulations of sympathetic noradrenergic neurons.

Role of Ca2+ in induction of neurotransmitter-related gene expression by butyrate.

We examined the effect of butyrate on neurotransmitter-related gene expression and calcium homeostasis in PC12 cells. Pretreatment with Ca2+ chelators (EGTA or BAPTA-AM) attenuated the butyrate-triggered accumulation of TH and ppEnk mRNA indicating that Ca2+ plays a role in butyrate-induced regulation of neuronal genes. Butyrate alone did not alter intracellular Ca2+ levels as determined by Fura-PE3 fluorescence; however, pretreatment with butyrate (18-24 h) reduced the first Ca2+ peak and prevented the second sustained rise in [Ca2+]i as induced by nicotine or ryanodine. In contrast, butyrate had no effect on Ca2+ transients when added shortly before or during nicotine or ryanodine stimulation. These results suggest that chronic butyrate exposure can modulate cell responses by affecting intracellular Ca2+ signaling.

Stereospecific regulation of tyrosine hydroxylase and proenkephalin genes by short-chain fatty acids in rat PC12 cells.

Circulating short-chain fatty acids (SCFAs) are primarily derived from bacterial fermentation of carbohydrates in the colon where they function as physiologic modulators of epithelial cell maturation. Butyrate has been shown to induce tyrosine hydroxylase, the rate-limiting enzyme of catecholamine synthesis, and enkephalin neuropeptide gene transcription, suggesting a role in perinatal sympathoadrenal stress-adaptation. We sought to determine whether there were SCFA structural requirements for this effect. Nine biologically relevant SCFAs and butyrate derivatives were tested in an in vitro model (PC12, rat pheochromocytoma cells) for their ability to regulate neurotransmitter-related gene expression. Our results revealed that among all the studied SCFAs, only propionate and butyrate increased tyrosine hydroxylase and proenkephalin mRNA levels. The functional activity was selective to the carbon atom chain length and associated with the presence of an ethyl moiety in the carbon atom backbone chain. Modifications or absence of this domain affected the gene induction response, suggesting a receptor-mediated mechanism(s). Moreover, propionate, butyrate, and the drug 4-phenylbutyrate were each shown to regulate transmitter genes via at least three independent mechanisms: histone hyperacetylation, cAMP signaling, or peroxisome proliferator-activated receptor gamma-mediated pathways. Thus, the biologic impact of SCFAs on catecholaminergic and opioid systems depend on the activation of SCFA-specific, dose-specific, and gene-specific molecular mechanisms. We speculate that 1) circulating levels of SCFAs may influence sympathoadrenal transmitter biosynthesis and hence whole animal stress-adaptive responsiveness after birth, and 2) the adverse effects of antibiotics on delayed acquisition of postnatal gut flora may affect this apparent evolutionary advantage of gut colonization.

Effect of haemorrhage on the expression of neurotransmitter-related genes in rat ventrolateral medulla: a quantitative real-time RT-PCR study

The ventrolateral medulla (VLM) has three functionally defined regions that contain catecholamine-synthesising neurons (rostral C1, caudal C1 and A1 regions). Many neuromessengers can alter cardiovascular functions in the VLM. The aims of this study were, first to validate the utility of real-time RT-PCR SYBR Green assay for quantitation of mRNA expression levels of neuromessengers in small site-specific neuronal populations in the VLM, and second to compare the basal mRNA levels of the adrenaline-synthesizing enzyme phenylethanolamine- N-methyltransferase (PNMT), neuropeptide Y (NPY) and preproenkephalin (ENK) in the three regions and third to examine the effects of haemorrhage on the expression of these three genes. Rats were anaesthetised with sodium pentobarbital and divided into three groups: perfused, sham-operated and haemorrhaged. A 15% haemorrhage was carried out on the haemorrhaged group. It was found that there are regional differences in the level of mRNA expression for all the three genes: with, in general, decreases from the rostral to caudal regions of VLM. A 15% haemorrhage significantly induced expression of PNMT in the rostral C1 region and NPY in the caudal C1 and A1 regions but had no effect on ENK at any sites, suggesting a differential regulation on the expression of these three genes in the VLM. Our results also demonstrate that real-time RT-PCR is a sensitive and accurate method for quantitative studies on neurotransmitter gene expressions in restricted brain regions.

High-Frequency Stimulation of the Subthalamic Nucleus Selectively Reverses Dopamine Denervation-Induced Cellular Defects in the Output Structures of the Basal Ganglia in the Rat

High-frequency stimulation (HFS) of the subthalamic nucleus (STN) is now recognized as an effective treatment for advanced Parkinson's disease, but the molecular basis of its effects remains unknown. This study examined the effects of unilateral STN HFS (2 hr of continuous stimulation) in intact and hemiparkinsonian awake rats on STN neuron metabolic activity and on neurotransmitter-related gene expression in the basal ganglia, by means of in situ hybridization histochemistry and immunocytochemistry. In both intact and hemiparkinsonian rats, this stimulation was found to induce c-fos protein expression but to decrease cytochrome oxidase subunit I mRNA levels in STN neurons. STN HFS did not affect the dopamine lesion-mediated overexpression of enkephalin mRNA or the decrease in substance P in the ipsilateral striatum. The lesion-induced increases in intraneuronal glutamate decarboxylase 67 kDa isoform (GAD67) mRNA levels on the lesion side were reversed by STN HFS in the substantia nigra, partially antagonized in the entopeduncular nucleus but unaffected in the globus pallidus. The stimulation did not affect neuropeptide or GAD67 mRNA levels in the side contralateral to the dopamine lesion or in intact animals. These data furnish the first evidence that STN HFS decreases the metabolic activity of STN neurons and antagonizes dopamine lesion-mediated cellular defects in the basal ganglia output structures. They provide molecular substrate to the therapeutic effects of this stimulation consistent with the current hypothesis that HFS blocks STN neuron activity. However, the differential impact of STN HFS on the effects of dopamine lesion among structures receiving direct STN inputs suggests that this stimulation may not cause simply interruption of STN outflow.

Alterations of neurotransmitter-related gene expression in human and experimental portal-systemic encephalopathy.

Portal-systemic encephalopathy (PSE) is a serious neuropsychiatric condition that results from chronic liver failure and portal-systemic shunting of venous blood. PSE is particularly prevalent following treatment of portal hypertension or ascites by the TIPS procedure. Recent studies both in autopsied brain tissue from PSE patients as well as in experimental animal models of PSE reveal that chronic liver failure results in altered expression of several genes coding for proteins having key roles in the control of neuronal excitability. Such alterations include increased expression of monoamine oxidase (MAO-A isoform), the "peripheral-type" benzodiazepine receptor (PTBR) as well as constitutive, neuronal nitric oxide synthase (nNOS). Such changes result in altered protein expression and in increased degradation of monoamine neurotransmitters, increased synthesis of neurosteroids with inhibitory properties and increased production of nitric oxide (respectively) in brain in chronic liver failure. In the case of PTBR and nNOS, increases in expression result from exposure to ammonia and/or manganese, two neurotoxic agents shown previously to be increased in brain in chronic liver failure. Further elucidation of the consequences of neurotransmitter-related gene expression could identify new pathophysiologic mechanisms and result in new approaches to the prevention of PSE in chronic liver disease in humans.

Neurotransmitter-related gene expression in intrastriatal striatal transplants. III. Regulation by host cortical and dopaminergic afferents.

Grafted striatal neurons have previously been shown to receive innervation from both the host cerebral cortex and dopaminergic substantia nigra. In the present study, we have used quantitative in situ hybridization histochemistry for striatal neuropeptide mRNAs, to determine the extent of functional integration exhibited by these two afferent systems. DARPP-32, preproenkephalin (PPE) and preprotachykinin (PPT) mRNAs were all expressed within discrete patches of the graft (termed P-regions) which corresponded well with each other on adjacent sections. Dopamine-depleting 6-OHDA lesions resulted in a marked increase in PPE mRNA levels and a concomitant decrease in PPT mRNA expression both in the remaining host striatum and in the P-regions of the graft. In a previous report [7], we have shown that cortical and dopaminergic afferents to the striatum interact in the regulation of PPE mRNA expression, such that in the absence of functional dopaminergic inputs, intact prefrontal corticostriatal afferents are necessary in order to maintain increased PPE mRNA levels. In the present study, we observed that cortical knife cut lesions placed at the level of the foreceps minor in previously 6-OHDA-lesioned animals resulted in a normalization of PPE mRNA expression, not only in the remaining host striatum but also within the P-regions of striatal grafts. Cellular analysis showed that this normalization was most pronounced in the peripherally situated P-regions (along the graft borders), which are known to receive dense host-derived cortical input. The cortical lesions had no significant effect on the 6-OHDA-induced reduction of PPT mRNA levels neither in the remaining lost striatum nor in the striatal graft. The expression of DARPP-32 mRNA in the remaining host striatum or striatal graft was not affected by either 6-OHDA lesion or cortical transection, demonstrating the specificity of the cortical lesion effect. These results indicate that both cortical and dopaminergic afferents originating in the host, functionally regulate neuropeptide mRNA expression within the striatal grafts, and that the two afferent systems interact with each other in the regulation of enkephalin gene expression in grafted neurons. On basis of recent results [9] showing that the enkephalin-expressing neurons are identical, at least in part, to efferent graft neurons projecting to the host globus pallidus, it is proposed that the cortical-dopamine interaction demonstrated here may play an important role in the recovery of complex motor performance induced by the striatal transplants.

Brain-derived neurotrophic factor and modify neurotransmitter-related gene expression in the 6-hydroxydopamine-lesioned rat striatum

Disruption of dopaminergic neurotransmission in the striatum by neurotoxic lesions of the substantia nigra leads to increases in glutamic acid decarboxylase and proenkephalin messenger RNA expression, and to decreases in preprotackykinin (the precursor molecule for substance P) messenger RNA expression in the two populations of striatal medium-sized spiny projection neurons. These cells also express TrkB, the neurotrophin receptor for brain-derived neurotrophic factor and neurotrophin 4/5, and TrkC, the receptor for neurotrophin-3. Since there is some indication that exogenous brain-derived neurotrophic factor can exert neuromodulatory effects in the basal ganglia, we studied the effects of repeated intrastriatal injections of the four members of the neurotrophin family of neural growth factors, nerve growth factor, brain-derived neurotrophic factor, neurotrophin-3, and neurotrophin-4/5 on the expression of striatal neurotransmitter-related genes in the unilaterally 6-hydroxydopamine-lesioned rat using in situ hybridization histochemistry. We found that 4 micrograms/day of brain-derived neurotrophic factor or neurotrophin-4/5 when injected intrastriatally for eight consecutive days led to a normalization of the denervation-induced decrease of preprotachykinin messenger RNA when compared to animals injected with equivalent doses of nerve growth factor, neurotrophin-3, or vehicle. Neurotrophin-4/5 alone also normalized expression of messenger RNA encoding the 67 x 10(3) mol. wt isoform of glutamate decarboxylase, while none of the neurotrophins had a significant effect on preproenkephalin messenger RNA expression.(ABSTRACT TRUNCATED AT 250 WORDS)